Project Summary Staphylococcus aureus (SA) accounts for 16% of all hospital-acquired infections (HAIs) and is responsible for more deaths in the U.S. annually than HIV/AIDS. The pathogen?s high morbidity and mortality are in part attributable to the fact that SA has developed resistance to many currently available antibiotics. Thus, there is an urgent need to discover/develop new therapeutic agents. Using an ocular staphylococcal infection models and an innovative genomic-based drug repurposing approach, we identified three drugs, Dequalinium Chloride (DC), Clofilium Tosylate (CT), and Glibenclamide (GLB), which can combat SA infection by modulating the genes/pathways activated during infection. Indeed, our in vitro preliminary studies revealed that both DC and CT exhibit direct bactericidal activities against SA and S. epidermidis (SE). Moreover, two drug combinations significantly reduced the expression of common inflammatory mediators, including IL-1? and TNF-?, in SA- challenged microglia, indicating the potential therapeutic effects of these drugs in staphylococcal infections. Studies outlined in the current proposal will evaluate the therapeutic efficacy of these novel drugs in vivo. In Aim-1, we will determine the therapeutic efficacy of novel drugs in mouse models of staphylococcal endophthalmitis. This will be accomplished by administering the drugs in two combinations, DC+GLB and DC+CT, at various times points post SA or SE infection in B6 mouse eyes. The drugs will also be tested in combination with the conventional antibiotic, vancomycin. The experiments will also examine the ocular toxicity of the drug combinations. Disease outcome will be measured by assessing bacterial burden, levels of inflammatory mediators, retinal tissue damage, and retinal function testing. Aim-2 is designed to investigate the molecular mechanisms underlying the therapeutic effects of the novel drugs and compare their modes of action with antibiotics. The proposed experiments include the administration of the best drug combination and a comparison of its therapeutic efficacy with that of vancomycin, followed by a comparative transcriptome and systems biology analysis of antibiotic vs. drug treated retinal tissue. Given the need for new anti-microbial therapeutic agents and the attractive features of drug repurposing, this study has considerable potential to develop novel therapies for both ocular and non-ocular staphylococcal infections.